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bioRxiv · 10.1101/2025.01.02.631138

Dissecting the Role of Flagellar Subunits in Clostridioides difficile Mucosal Colonization

Biswas, B.·Do, T. V. T.·Auchtung, J. M.·Piepenbrink, K. H.
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Abstract

Clostridioides difficile is a common cause of acute gastrointestinal (GI) inflammation in mammals, which can have detrimental effects on host health. C. difficile associated disease (CDAD) requires the secretion of high-molecular weight toxins after colonization of the GI tract. The molecular mechanisms of GI colonization by C. difficile, include potential interactions with host cells and the mucus layer formed from secreted mucin glycoproteins. C. difficile associates with the mucus layer in vivo and will associate with both epithelial cells and mucosal surfaces in vitro. Previously, we found a substantial defect in binding to mucosal surfaces for mutants of the major flagellar subunit, fliC, while mutation of the major subunit of type IV pili, pilA1, showed increased adhesion. To elucidate the mechanisms by which C. difficile interacts with ex vivo mucosal surfaces, we have measured swimming motility, mucosal adhesion and levels of flagellation by transmission electron microscopy for mutants of flagellar and T4P genes in C. difficile R20291. We discovered that the pilA1 mutant showed increased flagellation, while decreases in flagellation were found for fliC, fliD, and flg-OFF (a phase-locked mutant with low transcription of the F3 flagellar operon) which were associated with both low swimming motility and low adhesion to mucosal surfaces. However, the reversed flg-ON mutant showed increased flagellation without a significant increase in adhesion. We also found that the fliC mutant was defective in binding to mucus-secreting HT-29 MTX cells, but not HT-29 cells. These results imply that at least two molecular pathways contribute to C. difficile mucosal adhesion. In addition to their direct roles encoding T4P and flagellar subunits, pilA1 and fliC may contribute to regulating other factors relevant to mucosal adhesion.

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Biswas, B., Do, T. V. T., Auchtung, J. M., Piepenbrink, K. H.. 2025-01-03. Dissecting the Role of Flagellar Subunits in Clostridioides difficile Mucosal Colonization. https://doi.org/10.1101/2025.01.02.631138

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